The present invention is generally in the field of targeted therapeutic agents. By one of its embodiments, the present invention concerns an agent which specifically binds to receptors on certain cells. By a second embodiment the present invention concerns vectors specifically targeted to certain cells.
A specific aspect of the present invention concerns the prophylaxis and treatment of AIDS.
The following are references considered to be relevant for the subsequent description.
1. Salahuddin, S. Z., Ablashi, D. V., Markham, P. D., Josephs, S. F., Sturzenegger, S., Kaplan, M., Halligan, G., Biberfeld, P., Wong-Staal, F., Kramarsky, B., and Gallo, R. C. Isolation of a new virus, HBLV, in patients with lymphoproliferative disorders. Science, 234:596, 601, 1986.
2. Schirmer, E. C., Wyatt, L. S., Yamanishi, K., Rodriguez, W. J., and Frenkel, N. Differentiation between two distinct classes of viruses now classified as human herpesvirus 6. Proc. Natl. Acad. Sci., USA, 88:199-208, 1991.
3. Frenkel, N., Roffman, E., Schirmer, E. C., Katsafanas, G., Wyatt, L. S. and June, C. Cellular and growth factor requirements for the replication of human herpesvirus 6 in primary lymphocyte cultures, in: Immunology and Prophylaxis of Human Herpesvirus Infections, eds. Lopez, C., Mori, R., Roizman, B. and Whitley R. J., Plenum Publishing Corp. pp 1-8, 1990.
4. Kondo, K., Kondon, T., Okuno, T., Takahashi, M. and Yamanishi K. Human herpesvirus 6 infection of human monocytes/macrophages. J. Gen. Virol. 72:1401-1408, 1991.
5. Lusso, P., Ensoli, B., Markham, P. D., Ablashi, D. V., Salahuddin, S. Z., Tschachler, E., Wong-Staal, F., and Gallo, R. C. Production dual infection of human CD4+ lymphocytes by HIV-1 and HHV-6, Nature, 337:370-373, 1989.
6. Frenkel, N., Schirmer, E. C., Wyatt, L. S., Katsafanas, G., Roffman, E., Danovich, R. M., and June, C. H. Isolation of a new herpesvirus from human CD4+ T cells. Proc. Natl. Acad. Sci., USA, 87:748-752, 1990.
7. Wyatt, L. S., Rodriguez, W. J., Balachandran, N., and Frenkel, N. Human herpesvirus 7: antigenic properties and prevalence in children and adults. J. Virol., 65:6260-6265, 1991.
8. Wyatt, L., and Frenkel, N. Human herpesvirus 7 is a constitutive inhabitant of adult human saliva. J. Virol. 66:3206-3209, 1992.
9. Pellett, P. E., Lindquester, G. J., Feorino, P., and Lopez, C. Genomic heterogeneity of human herpesvirus 6 isolates. Adv. Exp. Med. Biol. 278:9-18, 1990.
10. Lawurence, P. J., Chee, M., Craxton, M. A., Gompels, U. A.,Honess, R. W., and Barrell, G. B. Human herpesvirus 6 is closely related to human cytomegalovirus. J. Virol. 64:287-299, 1990.
11. Lindquester, G. J., and Pellett, P. E. Properties of the human herpes-virus 6 strain Z29 genome: G+C content, length, and presence of variable-length directly repeated terminal sequence elements. Virology. 82:102-110, 1991.
12. Martin, M. E., Thomson, B. J., Honess, R. W., Craxton, M. A., Gompels, U. A., Liu, M. Y., Littler, E., Arrand, J. R., Teo, I., and Jones, M. D. The genome of human herpesvirus 6: maps of unit-length and concatemeric genomes for nine restriction endonucleases. J. Gen. Virol. 72:157-168, 1991b.
13. Frenkel, N., and Wyatt, L. S. Human herpesviruses 6 and 7 as exogenous agents in human lymphocytes. Develop. Biol. Standard. 76:259-265, 1992.
14. Lopez, C., Pellett, P. Stewart, J., Coldsmith, C., Sanderlin, K., Black, J., Warfield, D. and Feorino, P. J. Infect. Dis., 157:1271-1273, 1988.
15. DiLuca, D., Katsafanas, G., Schirmer, E., Balachanran, N. and Frenkel, N. Virology, 175:199-210, 1990.
16. Frenkel, N., Schirmer, E. C., Wyatt, L. S., Katsafanas, G., Roffman, E., Danowich, R. M. and June, C. H. Proc. Natl. Acad. Sci., USA, 87:748-752, 1990.
Human herpes virus-6 (HHV-6) was first isolated from peripheral blood mononucleur cells (PBMC) of patients with lympho proliforative disorders as well as from patients suffering from acquired immune deficiency syndrome (AIDS) (Salahuddin et al. 1986).
Two types of HHV-6 strains are recognized today and designated as variant A and variant B which differ as regards to their growth properties, restriction enzyme patterns and antigenicity and they are also distinct epidemiologically (Schirmer et al., 1991). Only the HHV-6 B variant appears to be associated with human disease and has been found to be the causative agent of exanthem subitum (ES, roseola infantum).
HHV-6 is shown to replicate only in interluken-2 (IL-2) activated T cells (Frenkel et al., 1990) and is inhibited by very high concentrations of IL-2. After the initial infection process, the HHV-6 virus undergoes a latency period in the infected cells (Kondo et al. 1991).
Recently, it has been shown that HHV-6 may effect the efficiency of expression of the human immunodeficiency virus-1 (HIV-1) when the two viruses have infected a single cell (Lusso et al., 1989).
Human herpes virus-7 (HHV-7) is a DNA virus first isolated in the laboratory of the inventor of the present invention from activated T cells expressing the CD4 antigen (see U.S. Ser. No. 07/553,798 and Frenkel et al., 1990). Cells expressing this antigen on their membrane will hereinafter be referred to as xe2x80x9cCD4+ cellsxe2x80x9d.
HHV-7 was found to be distinct, both molecularly and antigenically, from all previously identified herpes viruses. HHV-7 replicates well in lymphocytes and particularly in T cells including CD4+ T cells and possibly other cells carrying the CD4 marker. The HHV-7 virions specifically target the T cells wherein the viral DNA is synthesized in the nucleus as concatemers which are then cleaved and packaged into structural infectious particles. It has been shown recently that HHV-7 binds specifically to the CD4 receptor by which it infects CD4+ cells.
HHV-7 is found in sera of more than 95% of humans (Wyatt et al., 1991). In addition, the virus is very often found in human saliva (Wyatt et al., 1992). No known disease is associated with HHV-7 and no symptoms have been discovered in individuals infected by the virus at early childhood.
HHV-6 and HHV-7 DNA comprise a long unique sequence which is flanked by terminal direct repeats (TR) on each side (Pellet et al., 1990, Lawrence et al., 1990, Lindquester et al., 1991 and Martin et al., 1991). Each TR contains on one side a sequence which is heterogenous in size, designated het. The het sequence was thought to be a variable and unstable sequence but later was found to be a unique sequence for each virus strain and to remain stable in a single strain over many passages of the virus (Schirmer et al., 1991). On the other side of the TR there is a repeated telomeric-like sequence having repeated units of the sequence GGGTTA.
CD4+ cells are also the target cells of the human immunodeficiency virus (HIV) which is the cause of acquired immuno deficiency syndrome (AIDS).
HIV binds to the CD4 receptor on the target cell with a high affinity, integrates itself into the host cells"" genome and is believed to undergo a long latency period during which it is virtually undetectable. Activation of the virus to induce the disease may occur at different time periods after the first infection.
Many attempts have been aimed at delaying or inhibiting the activation of the latent HIV in an infected individual. Such attempts include various treatments targeted at inhibiting the HIV""s regulatory replication and structural proteins, e.g. Tat and Rev or by down regulation of these or other regulatory proteins of HIV. Examples of such treatments include the use of the drug AZT, both alone or in combination with other drugs such as ddI and nevipapine and methods of gene therapy by the use of an RNA virus vector.
In addition to treatments such as the above, research has centered mainly in trying to immunize individuals against infection or against a spread of HIV in the body of already infected individuals. For this purpose, both classic vaccination approaches as well as the use of various genetically engineered immunogens have been tested, but to date, none of these vaccination approaches have been found effective.
It is an object of the present invention to provide novel lymphotropic agents, i.e. agents capable of exerting therapeutic effects on lymphatic cells.
It is more specifically an object, in accordance with a first embodiment of the present invention, to provide a ligand capable of binding to the CD4 receptor (hereinafter: xe2x80x9cCD4-ligandxe2x80x9d). Such a ligand is useful for inhibiting the infectious process of viruses which infect the CD4+ cells and which enter the cells by first binding to the CD4 receptors. Another possible use of the CD4-ligand is as an immunomodulating agent.
It is more specifically an object in accordance with a second embodiment of the present invention to provide a DNA vector specifically intended for lymphatic cells (hereinafter: xe2x80x9clymphotropic vectorxe2x80x9d). Specific applications of the lymphotropic vector are in the treatment of AIDS as well as in the treatment of lymphatic malignancies, various autoimmune disorders, as well as a variety of T-cell pathologies.
Other objects of the present invention are the provision of pharmaceutical compositions and methods making use of the CD4-ligand or lymphotropic vector.
In accordance with a first embodiment of the present invention, there is provided a CD4-ligand, selected from the group consisting of:
(a) human herpes virus 7 (HHV-7);
(b) a mutant of HHV-7 capable of binding to the CD4 receptor;
(c) a virus particle of the virus of (a) or (b);
(d) a virion polypeptide of (c) capable of binding to CD4 receptor;
(e) a fusion protein of a fragment of (d) and another protein or peptide, which is capable of binding the CD4 receptor;
(f) derivatives of any of (c), (d) or (e) obtained by chemical modification, addition, deletion or replacement of one or more amino acid residues from the protein or peptides of (c), (d) or (e) which are capable of binding to the CD4 receptor; and
(g) any combination of the agents under (a), (b), (c), (d), (e) and (f).
In accordance with a second embodiment of the invention, there is provided a lymphotropic vector comprising a recombinant DNA molecule having:
(i) a DNA sequence derived from HHV-6 or HHV-7 and comprising an origin of DNA replication, a promoter sequence capable of inducing expression in a lymphatic host cell of a downstream nucleic acid sequence and a cleavage and packaging signal;
(ii) a foreign nucleic acid sequence downstream to an expression control of said promotor sequence.
For therapeutic use, said lymphotropic vector is incorporated into a delivery vehicle. A large number of vehicles are available for the delivery of genetic material into cells, delivery vehicle which are viral-derived particles are generally preferred in view of the specificity of such particles to certain cells which facilitate the targeting of the genetic material to such cells. Seeing that the lymphotropic vector of the invention is derived from HHV-6 or HHV-7, the preferred viral particle for use as a delivery vehicle is derived from these two respective viruses. There is some evidence that HHV-7 may activate HHV-6 replication (Frenkel et al. 1992), and accordingly, it is also possible in accordance with the invention to use an HHV-7 particle as a delivery vehicle for an HHV-6 derived lymphatic vector.
HHV-6 or HHV-7 particles have an affinity to specific cell types. The HHV-7, binds to the CD4 receptor and accordingly the particle derived from the HHV-7 is particularly useful for the delivery of said lymphotropic vector to CD4+ cells. The HHV-6 particles have an affinity to a variety of cells and mainly to both CD4+ and CD8+ cells, as well as to some other lymphatic cells, e.g. EBV infected B-cells, and may thus be useful for the targeting of said lymphotropic vector to such cells.
The preferred delivery vehicle in accordance with the present invention, is a member selected from the group consisting of:
(a) an HHV-6 or HHV-7 particle;
(b) a mutant HHV-6 or mutant HHV-7 particle capable of infecting lymphatic cells and delivering its content of DNA to such cells;
(c) a chemically modified particle of (a) or (b) essentially retaining the ability to infect lymphatic cells; and
(d) any combination of (a), (b) or (c).
It should be noted that the use made above and below of the term xe2x80x9clymphotropic vectorxe2x80x9d is only for convenience and does not mean to indicate that the vectors are limited only to lymphatic cells. As readily known, there are non-lymphatic cells which are CD4+, e.g. fibroblasts or various brain cells, and said vector, using HHV-7 as a delivery vehicle is thus useful also as a therapeutic agent targeted at such non-lymphatic CD4+ cells. Similarly, HHV-6 is known to be capable of infecting also non-lymphatic cells, e.g. fibroblasts, CD4+ brain cells, endothelial and epithilial cells and accordingly may be used as a therapeutic agent targeted also at such non-lymphatic cells.
Specific applications of said ligand or said lymphotropic vectors is in the treatment and/or prophylaxis of viral diseases which infect lymphatic, specifically CD4+ or CD8+, cells. An example of such an application is in the treatment and/or prophylaxis of HIV infections, in the treatment and/or prophylaxis of lymphomas or various autoimmune-related diseases or disorders, in the treatment of various T-cell pathologies, etc.
The present invention thus provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and, as an active ingredient, either said CD4-ligand or said lymphotropic vector. The present invention still further provides a method for the treatment of a disease or disorder comprising administering said CD4-ligand or said lymphotropic vector to an individual in need.
Said CD4-ligand may be used in order to inhibit infections of CD4+ cells which progress through the binding of the infectious agent to the CD4 receptor. A notable example of such is in the treatment of HIV infections. In addition, said CD4-ligand may also be used as an immuno-modulator, e.g. as a general immunosuppressor in autoimmune diseases.
Said lymphotropic vector is useful as an agent for genetic therapy in the treatment of various malignancies, viral infections, enzyme deficiencies and others, of lymphatic cells as well as other cells capable of being infected by HHV-6 or HHV-7. Two kinds of vectors are provided by the present invention: a vector which is capable of autonomous replication (hereinafter: xe2x80x9cARVxe2x80x9d (autonomously replicating vector)); a vector which is not capable of self replication (hereinafter: xe2x80x9cTampliconxe2x80x9d). While an ARV can be administered by itself, a Tamiplicon is administered together with a helper virus which provides the transactivation factors for replication of the Tamplicon. A helper virus is typically a self-replicating HHV-6 or HHV-7. The choice of the helper virus may typically be based on the nature of the Tamplicon: in case of a Tamplicon derived from HHV-6, a self-replicating HHV-6 will typically be used, and in the case of a Tamplicon derived from HHV-7, a self-replicating HHV-7 will typically be used. As pointed out above, a self-replicating HHV-7 may be used as a helper virus for an HHV-6 derived Tamplicon.
As already pointed out above, HHV-7 has no known pathology and therefore its use as a helper virus is generally preferred where possible over the use of HHV-6. However, use of HHV-7 is limited in view of the fact that it infects primarily CD4+ cells and accordingly use of HHV-6 is at times preferred. In case use is made of the HHV-6, measures should be taken to neutralize this virus after such period of time. Alternatively, a mutant HHV-6 may be used, the expression of which may be controlled by changes in various factors such as, a change in temperature (i.e. a temperature sensitive mutant).